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LWT - Food Science and Technology 65 (2016) 963e968

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LWT - Food Science and Technology

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Short communication

Alkaline phosphatase activity in cheese as a tracer for cheese milkpasteurization

Lotti Egger a, *, Marina Nicolas b, Luisa Pellegrino c

a Agroscope, Institute for Food Sciences, Schwarzenburgstr. 161, 3003 Bern, Switzerlandb National Reference Laboratory for Milk and Milk Products (EURL), Avenue du G�en�eral de Gaulle 23, 94706 Maisons Alfort Cedex, Francec Department of Food, Environmental and Nutritional Sciences (DeFENS), University of Milano, Via Celoria 2, 20133 Milano, Italy

a r t i c l e i n f o

Article history:Received 5 June 2015Received in revised form21 August 2015Accepted 18 September 2015Available online 25 September 2015

Keywords:Alkaline phosphatase activityPasteurization markerCheeseMilk

Abbreviations: ALP, Alkaline phosphatase.* Corresponding author.

E-mail address: charlotte.egger@agroscope.admin.

http://dx.doi.org/10.1016/j.lwt.2015.09.0330023-6438/© 2015 The Authors. Published by Elsevier

a b s t r a c t

Alkaline phosphatase (ALP) activity is used throughout the world as a marker for the proper pasteuri-zation of milk, to guarantee its hygienic safety. The Standard ISO 11816-2/IDF 155-2 describes the analysisof ALP in cheese. However, the method has been questioned in the past because there have sometimesbeen ambiguous results. The critical operations of the analytical procedure are more precisely definedand a zonal cheese sampling adopted. ALP inactivation is firstly evaluated in the relevant steps ofcontrolled cheese makings of hard (Emmental), semi-hard (Raschera) and soft (Chaource) cheeses.Application of the improved procedure in over 700 samples of typical cheeses from France, Italy andSwitzerland proved the applicability of the method. Based on this large study, a limit for ALP activity incheese from pasteurized milk is proposed at 10 mU/g.© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND

license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Alkaline phosphatase (ALP) activity is used throughout theworld as a marker for the proper pasteurization of milk, to guar-antee its hygienic safety (Rankin, Christiansen, Lee, Banavara, &Lopez-Hernandez, 2010). Milk pasteurization is also adopted inmanufacturing of several cheese varieties with the aim ofdestroying the possible pathogenic microorganisms and reducingthose capable of causing defects in the cheese (Kousta, Mataragas,Skandamis, & Drosinos, 2010). Heat-treatments milder thanpasteurization are sometimes used to achieve a reduction of un-desirable species while preserving the ability of milk to coagulate.At small dairies, milk is often thermally treated using poorlycontrolled batch processing. In addition, some countries poserestrictions to production or import of cheeses produced fromunpasteurized milk (Pellegrino & Donnelly, 2004; Ryser, 2011).Considering all these situations, a simple and reliable method tocheck proper milk pasteurization in cheese would be of high valuefor both safety inspection and trading controls. The analysis of ALPin cheese is described by ISO 11816-2/IDF 155-2 (ISO, 2015). In the

ch (L. Egger).

Ltd. This is an open access article u

past, this proposed method was not considered appropriate forreflecting the heat treatment of the cheese milk in some types ofcheese (Lechner & Ostertag, 1993). Further studies evidenced thatthe high variability of different cheese varieties in processingconditions, texture and size affects the residual activity of ALP andits zonal distribution within the cheese (Bisig, Frohlich-Wyder,Jakob, & Wechsler, 2010; Pellegrino, Tirelli, Masotti, & Resmini,1996). The temperature at which the curd is heated and the size ofthe cheese wheel are the main parameters influencing residualALP activity in cheese. Therefore, besides of having a reliableanalytical method, there is an urgent need for an appropriate limitfor residual ALP activity to characterize cheeses from pasteurizedmilk. This work performs an in-depth study of the relevanttechnological parameters for cheese making in order to define thecritical analytical conditions and possible limitations on how ALPactivity in cheese can be used as a marker for the pasteurization ofcheese milk. In addition, a survey of the ALP levels in commercialcheeses has been performed to confirm the feasibility of theproposed approach.

2. Materials and methods

2.1. Cheese production and sampling

Cheeses selected for the study, i.e. Emmental (hard cheese),

nder the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

L. Egger et al. / LWT - Food Science and Technology 65 (2016) 963e968964

Raschera (semi-hard) and Chaource (soft cheese), were manufac-tured under controlled conditions at respective dairies, and sam-ples were taken at different steps of the process. The usualprocessing conditions were followed in all cases, and the use ofeither raw, thermized (63e67 �C for 15e30 s) or pasteurized(73e78 �C for 20e30 s) milk were the only variables adopted in theprocess. The cheeses were ripened under the usual conditions, andsamples were taken at the end of the commercial ripening period,at 4 months, 2 months and 15 days, for Emmental, Raschera andChaource cheeses, respectively.

Commercial cheese samples from France, Italy andSwitzerland were classified into three different groups relating tocheese type (soft, semi-hard and hard cheese) and milk thermaltreatment (unheated, thermized, pasteurized), according to theproduct information. For cheeses produced under controlledconditions, samples for zonal measurements of ALP were takenin the different zones, as shown in the Section 3.2 (Fig. 1C).Commercial cheeses were sampled according to the schemeproposed in Section 3.3. From each cheese, a representativeportion of the corresponding gray zone (Fig. 2) was grinded andhomogeneously mixed.

2.2. Analysis of alkaline phosphatase with fluorophos

Milk samples were analyzed according to ISO 11816-1/IDF 155-1(ISO, 2013), while the cheese samples were analyzed based on ISO11816-2/IDF 155-2 (ISO, 2015), with some optimizations.

Briefly, cheese buffer (Fluorophos Test System, Advanced In-struments Inc., Norwood MA, USA) was used for cheese extrac-tion instead of milk. An aliquot of 0.3e0.5 g of the grindedcheese was weighed in a glass beaker, 5 mL of cheese buffer wasadded and then this was mixed with a homogenizer (e.g., UltraTurrax®) to obtain a completely homogenous dispersion. Aftertransferring the sample to a 25 mL volumetric flask, the glassbeaker was rinsed with 5 mL of cheese buffer and mixed onceagain with the homogenizer. The samples were pooled, and theflask was filled to the mark with cheese buffer. The sample wascentrifuged at 1000 g at 4 �C for 10 min. The middle transparentphase was collected in a clean tube, and 75 ml were taken foranalysis according to ISO 11816-2/IDF 155-2 (ISO, 2015), asindicated above.

2.3. Statistical analysis

At least 150 individual samples were analyzed in duplicate foreach cheese type. The data were analyzed for variance by pairwisecomparison with the Fisher's 95% Least Significant Difference posthoc test, using least squares means with SYSTAT 13 (Systat Soft-ware, Inc., Chicago, IL, USA). Values of P < 0.001 were considered tobe significant.

3. Results and discussion

3.1. Impact of cheese milk processing on ALP activity

The batches of milk that were used for making the controlledcheeses were analyzed for ALP activity according to the preparationsteps. The samples for either raw, thermized or pasteurized milkwere taken after milk fat standardization (not done for Rascheracheese), maturation at low temperature (10e16 �C) and heating tothe temperature required for coagulation (32e38 �C). Data from themanufacturing process for Emmental cheese are shown in detail, as

an example, in Fig. 1. As expected, the ALP activity in the differentlyheated milk samples decreased with treatment at higher temper-atures (Fig. 1A). The subsequent steps did not have any furtherimpact on the ALP activity. Very similar behavior was observed forthe other two cheeses (not shown).

3.2. Zonal differences of ALP inactivation in cheese

To study the effects of the heat treatment during cheeseproduction on the resulting zonal differences in heat load in thewhole cheese, different types of cheeses were manufacturedunder controlled conditions with the previously differentlyheated milk.

It was already demonstrated that in large-size cheeses, suchas Grana Padano, Parmigiano-Reggiano and Emmental cheese,ALP is inactivated within the cheese loaf during molding becauseof the high temperature and the low pH conditions that remainfor several hours. Since these cheeses are made with raw milk,residual ALP activity is only present in the peripheral portion ofthe cheese that cools down faster (Bisig et al., 2010; Pellegrinoet al., 1997). In this study, Emmental cheese was chosen asmodel because of its large-size wheel deriving from the tradi-tional curd pressing process. As for the other cheeses that havebeen mentioned, the curd is cooked at high temperature(53e56 �C), which has allowed us to study the zonal differencesin ALP activity in the ripened cheese depending on preliminarymilk heat treatment.

After the cheese had ripened for four months, four differentsamples were collected from the cheese wheel (Fig. 1 B) andanalyzed for ALP activity. One sample was representative of thewhole cheese (Fig. 1 B, Whole), while three were zonal samples(Fig. 1 B, C) taken from the outer, intermediate and core zones. Asexpected, ALP activity was highest for all milk heat treatments inthe outer zone and decreased towards the core zone of thecheese loaf (Fig. 1 B). ALP activity in the whole cheese samplewas clearly less than the activity of the outer zone. Also,although the differences in the milk heat treatment could beobserved in all the samples, they were the greatest in the outerzone. In all three zones, the heat treatment at 73 �C for 20 s,corresponding to milk pasteurization conditions, was found tolead to ALP activities below 10 mU/g (Fig. 1 B). However, in thecore zone, the differences in residual ALP activity betweentreatments at 67 �C/20 s and 73 �C/20 s were very small. Thisindicates that a distinction between pasteurization and therm-ization (Ryser, 2011) is not possible in samples from the corezone of the cheese.

In the smaller cheese wheels of both Chaource (soft cheese) andRaschera (semi-hard), the zonal differences were marginal (datanot shown) and, therefore, a zonal sampling in those cheeses didnot improve the results. In the cheeses produced with pasteurizedmilk, the ALP activity was below 10 mU/g, irrespective of thesampling procedure.

3.3. Recommended cheese sampling

According to the data that was obtained in this study and re-ported previously, zonal differences in ALP activity are expected inhard cheeses with big wheels and relatively high curd cookingtemperatures. The typical raw milk cheeses from Italy, Switzerlandand France, such as Emmental, Grana Padano, Parmigiano-Reggiano, Gruy�ere, Sbrinz and Bernese Hobel cheese, belong tothis group of cheeses. As a result of the zonal differences that were

Fig. 1. ALP activities of milk after different heat treatments and of the derived Emmental cheeses. ALP activities of cheese milk following different heat treatments and taken atdifferent steps of the preparation process. Error bars represent standard deviation of duplicated analyses (A). ALP activities measured in different zones of the derived cheeses (B).Sampling zones considered (C).

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Fig. 2. Cheese Sampling. Gray areas indicate the proposed sampling zones for HardCheeses (A), Semi-hard Cheeses (B) and Soft Cheeses (C).

L. Egger et al. / LWT - Food Science and Technology 65 (2016) 963e968966

observed in the hard cheeses, a specific sampling scheme wasestablished (Fig. 2). According to this scheme, hard cheeses shouldonly be sampled from the outer zone (A), semi-hard cheesesshould be sampled from the entire portion (B) and soft cheesesshould be sampled from two opposite quarters of the whole wheel(C). The sampling scheme for semi-hard and soft cheeses corre-sponds to the ISO 707/IDF 50 Standard (ISO, 2012), while thesampling zone for hard cheeses has not previously been described.In some cases, the assignment of a cheese variety to hard or semi-hard cheese type can be difficult. In Codex Alimentarius (2013) thedesignation of cheeses according to their firmness is based on themoisture content on a fat-free basis, but the values limiting thetwo classes partially overlap. Nevertheless, our results demon-strate that sampling a semi-hard cheese using the methoddescribed for hard cheeses will not result in different values of ALPactivity. Moreover, the sampling for hard cheeses can only beperformed on cheese samples where the cheese rind is presentand clearly visible, which excludes the analysis of small pieces orgrated cheese. Cattaneo et al. (2008) demonstrated that, using asuitable extraction procedure, ALP can be determined in gratedGrana Padano cheese. However, grated cheeses only represent alow proportion of commercial cheeses and were not considered inthis study.

3.4. ALP activities in pasteurized, thermized and raw milk cheeses

More than 700 samples of 32 different cheese varieties orig-inating from France, Italy and Switzerland were collected,sampled according to the above proposed sampling scheme andanalyzed for ALP activity. Cheeses made from pasteurized, ther-mized and raw milk were considered. All the average values foreach cheese variety that was measured, including the standarddeviation (SD) and number of analyzed samples (N) are compiledin Table 1. The high SD values for certain types of cheese made

from raw milk can be attributed to a number of factors, mainlydifferences in the cheese-making conditions and in the ripeningtime. It is also important to consider that many of those cheesesare manufactured at artisanal dairies. Among the raw milkcheeses, the lowest values of ALP activity were found in GranaPadano and Parmigiano-Reggiano cheeses, which are ripenedover a period of 12 months at least. In fact, ALP was shown todecrease progressively in these cheese types during the pro-longed ripening and to reach approximately 300 mU/g after 34months (Pellegrino et al., 1996). In contrast, the high valuesfound in the Bernese Hobel cheese could be related to a lowercurd heating temperature compared to other hard cheeses and tothe smaller cheese mold. However, it is important to note that allthe pasteurized cheeses have ALP activities that are clearly below10 mU/g. This indicates that a clear-cut decision can be made if aspecific cheese has been produced from pasteurized milk whenthe proposed sampling scheme is respected.

Individual statistical analysis of the measured ALP activities forsoft (A), semi-hard (B) and hard (C) cheeses showed that, within thethree cheese types, three different processing of cheese milk:pasteurization, thermization and no heat treatment (raw milk),were all significantly different from each other (P < 0.0001) in theirALP activities (Fig. 3).

3.5. Tentative limit for pasteurized cheese

A clear-cut difference between cheeses from pasteurized milkand cheeses from thermized or raw milk can be drawn at the levelof 10 mU/g. To our knowledge, very few data available from theliterature was obtained with the same analytical method and is,therefore, comparable to ours. Hodoscek, Rupnik, Ahcin, andBiasizzo (2012) report ALP activity values in the range of0.5e3.2 mU/g in 19 samples of Slovenian cheeses produced frompasteurized cow milk and including hard, semi-hard and soft va-rieties. Values of ALP were higher than 400 mU/g in cheeses madefrom either raw or thermized milk. A recent survey of ALP activityin ten representative cheeses made from pasteurized milk in theUnited Kingdom indicates levels ranging from 0.1 to 4.6 mU/g(Cassidy, Daly, Early, & Rowe, 2014).

To date, only two cheese types have been found for which themethod has not been applicable. Blue cheeses need to be excludedbecause of the activity of the phosphatases from molds (usuallyfrom Penicillium spp. and Aspergillus spp.) (Rosenthal, Bernstein, &Rosen, 1996). In addition, the temperature over 60 �C reached bythe curd for pasta-filata cheeses, while is being stretched with hotwater (Kindstedt, Caric, & Milanovic, 2004), inactivate ALP makingit impossible to test whether the cheese milk was correctlypasteurized.

4. Conclusions

Alkaline phosphatase activity is a pertinent marker for properpasteurization of the cheese milk if the proposed cheese sampling,especially for hard cheeses, is respected and with exclusion of bluecheeses and pasta filata cheeses. Based on data obtained from over700 individual samples from three different countries, a tentativelimit for the ALP values of pasteurized cheeses can be set at 10 mU/g. The reliability of this limit is confirmed by literature data.Nevertheless, further work on cheeses from other countries will beperformed to support and confirm the applicability of the methodas well as the tentative limit of 10 mU/g.

Fig. 3. ALP activities of different cheese types produced with either raw, thermized or pasteurized milk. Whisker plots of ALP activities (log mU/g) measured in Soft, Semi-hard andHard Cheeses made either from pasteurized (Past), thermized (Therm) and raw milk, respectively. Box plots show median, length of box represents 50% of the central values, andasterisks represent values outside the inner fence (>1.5 times outside the box hinge). Within each box, all three groups are different (P < 0.0001). Figures in brackets indicate thenumber of samples. A tentative limit of 10 mU/g for cheeses made from pasteurized milk is indicated.

Table 1Compiled results of ALP activities measured in typical cheeses mainly from France, Italy and Switzerland. SD¼ Standard deviation, N ¼ number of cheeses analyzed in du-plicates. Values written in gray color: analysis of single cheese samples in duplicate (N ¼ 1).

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Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

We thank Anne-C�ecile Boitelle and Caroline Desbourdes fromthe EURL, Veronica Rosi from the University of Milan, AndreasSpahni, Mirjam Abboud and Brigit Messerli from Agroscope for theanalysis of cheeses. Thanks for the support by the Centre NationalInterprofessionnel de l'Economie Laiti�ere (CNIEL), the French Na-tional Dairy Association, and Mr Jean-Pierre Guyonnet and MrMichel Place.

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